This invention is in the field of organic chemistry, in particular relating to methods for forming amide bonds useful for the ligation of molecules to surfaces.
Microarrays in which proteins or other compounds are immobilized to a surface enable high-throughput experiments that require only small amounts of analyte. Such protein “chips”, for example, can be used to detect protein-ligand, protein-protein, and antibody-antigen interactions. Attaching proteins or other compounds covalently, rather than noncovalently produces more robust surfaces. Attaching compounds to a surface in a uniform rather than random manner and in a manner which results in high-density attachment can provide a substantial advantage in assay sensitivity.
A useful method of forming amide bonds in peptide synthesis is through Staudinger ligation. In the Staudinger reaction a phosphine is used to reduce an azide to an amine:PR3+N3R′+H2O→O═PR3+H2NR′+N2(g).The intermediate in the reaction is an iminophosphorane (R3P+—−NR′), which has a nucleophilic nitrogen. Villarassa and others have shown that this nitrogen can be acylated, both in intermolecular and intramolecular reactions. (For examples, see: Garcia, J.; Urpí, F.; Vilarrasa, J. Tetrahedron Lett. 1984, 25, 4841-4844; Garcia, J.; Vilarrasa, J. Tetrahedron Lett. 1986, 27, 639-640; Urpí, F.; Vilarrasa, J. Tetrahedron Lett. 1986, 27, 4623-4624; Bosch, I.; Romea, P.; Urpí, F.; Vilarrasa, J. Tetrahedron Lett. 1993, 34, 4671-4674; Inazu, T.; Kobayashi, K. Synlett. 1993, 869-870; Molina, P.; Vilaplana, M. J. Synthesis-Stuttgart 1994, 1197-1218; Bosch, I.; Urpí, F.; Vilarrasa, J. J. Chem. Soc., Chem. Commun. 1995, 91-92; Shalev, D. E.; Chiacchiera, S. M.; Radkowsky, A. E.; Kosower, E. M. J. Org. Chem. 1996, 61, 1689-1701; Bosch, I.; Gonzalez, A.; Urpí, F.; Vilarrasa, J. J. Org. Chem. 1996, 61, 5638-5643; Maunier, V.; Boullanger, P.; Lafont, D. J. Carbohydr. Res. 1997, 16, 231-235; Afonso, C. A. M. Synthetic Commun. 1998, 28, 261-276; Tang, Z.; Pelletier, J. C. Tetrahedron Lett 1998, 39, 4773-4776; Ariza X.; Urpi, F.; Viladomat, C.; Vilarrasa J. Tetrahedron Lett. 1998, 39, 9101-9102; Mizuno, M.; Muramoto, I.; Kobayashi, K.; Yaginuma, H.; Inazu, T. Synthesis-Stuttgart 1999, 162-165; Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc. 1999, 121, 284-290; Boullanger P.; Maunier, V.; Lafont, D. Carbohydr. Res. 2000, 324, 97-106; Velasco, M. D.; Molina, P.; Fresneda, P. M.; Sanz, M. A. Tetrahedron 2000, 56, 40794084; Malkinson, J. P.; Falconer, R. A.; Toth, I. J. Org. Chem. 2000, 65, 5249-5252.) Hydrolysis of the resulting amidophosphonium salt gives an amide and phosphine oxide.
Saxon and Bertozzi have shown that the phosphine can also serve as the acyl donor. (Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010; Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Left. 2000, 2,1939-1941) Saxon et al. have reported a modification of the Staudinger ligation to form an amide from an azide using a phosphine reagent. (Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 2141-2143.) The phosphine reagents:
when reacted with an azidonucleoside are reported to result in the formation of an amide by acyl group transfer. The ligation is called “traceless” because no portion of the phosphine reagent other than the acyl group remains in the product.
The Staudinger ligation can be used to couple a peptide with no residual atom that remains in the amide product. (See U.S. patent application Ser. No. 10/456,988 filed Jun. 6, 2003, PCT Application PCT/US01/15440 filed May 11, 2001 and published Nov. 22, 2001 as WO 01/87920 A2, Poster presented at the ACS meeting in New Orleans, La. on Mar. 24, 2003 and at the National Organic Symposium in Bloomington, IN on Jun. 1, 2003. See also Nilsson et al. J Am Chem Soc. 2003, 125(18), 5268-5269; Soellner et al. J Org Chem 2002 67(14), 4993-4996; and Nilsson et al. Org Left. 2001 3(1), 9-12.) A protein, peptide (or fragment thereof) having a C-terminal phosphinothioester reacts with another protein, peptide (or fragment thereof) having a N-terminal azide. The resulting iminophosphorane leads, after an S- to N-acyl shift, to an amidophosphonium salt, where the P—N bond is hydrolyzed spontaneously to produce an amide (See Scheme 1).